PROJECT SUMMARY The proposed work would continue a collaborative effort aimed at identifying and mechanistically characterizing new molecular strategies for combating pathogenic bacteria. Antibiotic drugs for systemic use receive the most attention in this realm. In contrast, this work focuses on novel materials that could act at interfaces between the human body and the external world. The approach, inspired by natural host- defense strategies, features an unusual combination of synthetic and novel analytical tools. Humans and other multicellular organisms deploy small antimicrobial peptides (AMPs) to protect interfaces with the external world, such as skin and the GI tract, from pathogenic bacteria. AMPs exert broad-spectrum antibacterial activity. One common mechanistic theme involves disruption of bacterial membranes. Microbes evolve resistance to this mode of action only very slowly. AMP-inspired peptides are currently in clinical trials or under consideration for treatment of diabetic foot ulcers, nasally colonized pathogens, middle ear infections, GI tract infections, lung infections, wound sterilization, and prevention of colonization of implanted devices (e.g., catheters). However, the cost of production of sequence-specific peptides is problematic for many biointerface-based applications. This research program grew out of the unconventional hypothesis that AMP activity might not require a defined subunit sequence. We have shown that sequence-random copolymers can mimic the activity profile characteristic of AMPs. Nylon-3 polymers are the focus because their protein-like backbone (?-amino acid- derived subunits) promotes biocompatibility, and because considerable structural diversification can be achieved. Generation of the active materials via a polymerization process is much less expensive than the step-by-step synthetic methods that are necessary to produce sequence-specific peptides. This proposed effort includes synthesis and evaluation of many new nylon-3 copolymers with widely varying characteristics, with the goal of optimizing biological activity profiles and laying a foundation for biomedical applications. The copolymers comprise a mixture of hydrophilic and hydrophobic subunits. Unexpected results from recent studies of hydrophobic subunit variation lead us to propose a parallel study of cationic subunit variation. Our distinctive single-cell measurements will be adapted to allow us to survey large numbers of new polymer samples. A new aspect of the program will use solid-phase polypeptide synthesis in a novel way to prepare subsets of the diverse chain populations generated via polymerization reactions. Assessing these submixtures by conventional and single-cell methods should provide unprecedented insight into relationships between polymer structure and biological activity. This work will deepen our mechanistic understanding of nylon-3 polymer activity and ultimately lead us to new polymer compositions that manifest improved antibacterial activity and prokaryotic vs mammalian cell selectivity.